At first glance, it seems that nothing is new in the cultivation of Mycobacterium tuberculosis. The first attempts of M. tuberculosis cultivation on agar medium go back to the report by Fannie and Walter Hesse in 1881. In 1882, Robert Koch used blood serum coagulated on glass slides for M. tuberculosis cultivation. Apparently, he was not too much concerned about the biosafety of such a procedure. He later improved this method, which was called the “plate technique”, by adding peptone, some salts and glycerol. Also, in 1882, Richard Petri invented the petri dish to be used instead of a glass slide. These attempts at cultivation on a transparent type of media were interrupted in 1903 with introduction of the first egg-based media by Dorset (Dorset, Science. 17:374, 1903), followed by a variety of egg-based media recipes (American Trudeau Society, Handbook of Tuberculosis Laboratory Methods, Washington, D.C., 1962; IUAT, Bull Int Union Tuberc Lung Dis. 24:78, 1954; Jensen, Abteilung Originale. 125:222-239, 1932; Ogawa et al., Kekkaku. 24:13-29, 1949; Petragnani, Bollettino dell'Istituto sieroterapico Milanese. 5: 173-185, 1926; Petroff, J. Exp. Med. 21:38-42, 1915; Stonebrink, Acta Tuberc. Scand. 35:67-80, 1958).
Based on systemic studies of the tubercle bacilli requirements, Dubos and Middlebrook in 1947 introduced the first effective agar medium under the name “Dubos Oleic Acid Albumin Agar” (Dubos, Amer. Rev. Tuberc. Pulm. Dis., 56:334-345, 1947). It followed by an invention by Middlebrook and Cohn of the 7H10 agar in 1958 (Middlebrook et al., Amer. J. Publ. Health, 48:844-853, 1958) and 7H11 agar in 1968 (Cohn et al., Am. Rev. Respir. Dis. 98:295-296,1968). Subsequently, two recipes of a selective agar medium containing four antimicrobials to prevent the growth of contaminants (Polymyxin B, Amphotericin B, Carbenicillin, Trimethoprim) were introduced in 1972 (Mitchison et al., J. Med. Microbiol. 5:165-175, 1972) and in 1976 (McClatchy et al., Am. J. Clin. Pathol. 65:412-415, 1976). A powder base for all these media, 7H10 agar, contains agar, combination of seven salts, L-glutamic acid, pyridoxine, biotin, malachite green. It is available commercially from a number of manufacturers. Preparation of an agar medium requires addition of the OADC growth supplement, which contains sodium oleate, albumin (bovine, fr. V) dextrose, catalase.
At the time the 7H10 and 7H11 agar media were developed by Middlebrook and his colleagues working at the National Jewish Hospital in Denver, Colo., attempts to use an animal serum as a nutritional supplement failed because of inconsistent results with different serum batches. Therefore, a combination of the only standardized products available at that time, sodium oleate with bovine albumin, were used as the major component of the OADC supplement. The role of the bovine albumin is to neutralize the oleic acid, which is toxic to mycobacteria, in case of breakdown of sodium oleate. This composition required a final pH of 6.8, in spite of the known fact that the optimal pH for M. tuberculosis is 6.2-6.4. Subsequently, the presence of 5-7% of CO2 in the incubator is required to protect the growing mycobacteria from further alkalization of the environment due to the release of ammonia by the growing mycobacteria. Until the time of the present invention, these culture conditions remained the standard in the art.
The agar-based medium developed by Middlebrook and colleagues has a number of advantages over egg-based medium. One of them is transparency, which enables earlier detection of growing colonies. Among the major disadvantages of the existing agar media are the cost, difficulties in standardization of the OADC supplement, and the need for a CO2 incubator. These disadvantages represented the main obstacle in implementing the agar media outside of the US, especially in developing countries.
Timely identification of patients with drug resistance, especially those with multi-drug resistant tuberculosis (MDR-TB), is a key element of any comprehensive program targeting prevention of epidemics of drug-resistant tuberculosis. Drug susceptibility testing of the patients' isolates as a means to address this problem in developing countries has been considered impractical and unrealistic. A number of reasons for this, real or fictional, have been given by different authors, such as extreme sophistication of the procedure, high cost, and extended turnaround time for laboratory reports.
Pyrazinamide (PZA) is one of the first-line drugs in the standard treatment regimen currently used for tuberculosis patients. It is now mandatory in the United States (man. 12), and it is desirable for any country with high prevalence of drug resistance (man. 3) to perform a drug susceptibility test for pretreatment isolates from all new tuberculosis patients, at least with the first-line drugs. A variety of techniques can be used for susceptibility testing with any of the anti-tuberculosis drugs other than PZA, including the agar proportion method in its direct or indirect versions. However, the only approved test using PZA in the United States is the radiometric method using the special PZA liquid medium with pH 6.0 in the BACTEC-460 system (man. 10). This method is quite expensive and may not be affordable for many laboratories, especially in developing countries. In addition, the method requires the disposal of a substantial volume of 14C-radiolabeled culture vials, which is not permitted in many countries. Further, the test is not fully standardized and may give false-positive and false-negative results (man. 4-6). Unlike the agar proportion method test for other anti-tuberculosis drugs, the test in a liquid medium does not provide any information on the actual proportion of the PZA-resistant bacteria in the patient's bacterial population, and it cannot be used as a direct test (i.e., a pure culture must be isolated from the patient sample prior to testing). Therefore, the total turnaround time of the method is about four weeks, at best. There has been a suggestion to use the agar proportion method for PZA by substituting an ADC supplement for the conventional OADC supplement in a pH 5.5 agar medium (man. 1, 2). However, this revised method did not find its way into the clinical laboratory practice because of very poor growth of Mycobacterium tuberculosis isolates a pH 5.5 in the conventional agar medium (man. 11). Therefore, while a number of alternative methods for susceptibility testing with other drugs are widely available, the BACTEC method remains the only reliable technique for a test with PZA. Moreover, PZA only works at an acidic pH, which alone can inhibit the growth of M. tuberculosis on conventional medium.
Therefore, prior to the present invention, there was a need for a new agar medium, which does not require sophisticated or expensive cultivation techniques, which can be produced at a lower cost, and which can be used for susceptibility/resistance screening of Mycobacterium tuberculosis isolates against a wide range of drugs, including Pyrazinamide, with a reasonable turnaround time.